Fluorophores are molecular components that cause molecular fluorescence. Fluorophores absorb light energy having one wavelength and emit light energy at a different wavelength. The wavelengths of the absorbed light and the emitted light are commonly and respectively referred to as the excitation wavelength and the emission wavelength. The difference between the wavelengths of the absorbed light and the emitted light is referred to as the Stokes shift. The emission wavelength and the intensity of the emitted energy may depend, for example, on the nature of the fluorophore itself and the surrounding chemical environment.
Researchers may use spectrofluorometers to analyze the fluorescence characteristics of molecular fluorophores. Fluorescence characteristics may include, for example, the intensity of the emitted energy, fluorescence polarization, fluorescence lifetime, and time-resolved fluorescence. Researchers may also analyze the fluorescence characteristics of fluorophores that undergo spectral changes due to varying assay conditions, for example, changes to the intrinsic fluorescence of proteins that depend the three-dimensional folding of the protein.
Spectrofluorometry may involve spectral optimization to identify the excitation-emission wavelength pair that results in the optimal sensitivity level for the fluorophore. Conventionally, researchers may manually set the excitation-emission wavelength settings of the spectrofluorometer to measure fluorescence intensity and manually increment the settings through a desired wavelength range. This approach may be time-consuming and can be prone to error. Further, if a researcher has no prior knowledge of the fluorophore, the researcher may be required to perform a scan across the entire wavelength range in order to identify a wavelength range of interest.
In some situations, researchers may inappropriately select an excitation bandpass and an emission bandpass that are too close together. If the excitation and emission bandpasses are set too close together, crosstalk may result from excitation light leaking into the emission channel. In other circumstances, researchers may set the excitation-emission bandpasses such that the maximum raw intensity results. In either case, the perceived optimal excitation-emission wavelength pair may not result in the optimal sensitivity level for the fluorophore.
Therefore, a need exists for automatically determining the optimal excitation-emission wavelength pair of a fluorophore that results in the optimal sensitivity level for the fluorophore.